The annealing of tRNA3Lys to human immunodeficiency virus type 1 primer binding site is critically dependent on the NCp7 zinc fingers structure.
The nucleocapsid protein NCp7 of the human immunodeficiency virus type 1 contains two zinc fingers of the CX2CX4HX4C type, flanked by several basic residues, and plays a major role in viral infectivity. Thus, NCp7 was shown to promote annealing of the tRNA3Lys to the primer binding site, a key step in reverse transcription. However, previous in vitro experiments were unable to clarify the role of the zinc fingers in this process, due to nucleic acid aggregation induced by the basic N- and C-terminal domains of NCp7. We show here that deletion of these sequences in (12-53)NCp7 strongly reduces the formation of aggregates and allows a direct visualization of the binary or ternary complexes between NCp7 and nucleic acids by gel electrophoresis. (12-53)NCp7 is able to induce hybridization of the 33P tRNA3Lys and the human immunodeficiency virus type 1 viral RNA-(77-257), which contains the primer binding site. Modification of the proximal zinc finger conformation in Cys23(12-53)NCp7 led to a large reduction in this hybridization process, while replacement of Trp37 by Leu in the distal zinc fingers resulted in a complete absence of annealing activity. These data account for the in vivo loss of viral infectivity following these mutations and emphasize the critical role of the structure of the zinc finger domain of NCp7. This could facilitate a rational approach to new antiviral agents directed toward NCp7.
Remy
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E
Département de Pharmacochimie Moléculaire and Structurale, U266 INSERM-URA D1500 CNRS, UER des Sciences Pharmaceutiques et Biologiques, 4, Avenue de l'Observatoire, 75270 Paris Cedex 06, France.
de Rocquigny
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H
Petitjean
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P
Muriaux
D
D
Theilleux
V
V
Paoletti
J
J
Roques
B P
BP
D000595:Amino Acid Sequence; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D012357:RNA, Transfer, Lys / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins; D016335:Zinc Fingers / Q000235:genetics; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus